The unicellular parasite Trichomonas vaginalis is responsible for the most prevalent, non-viral, sexually-transmitted infection worldwide, with approximately 1/4 billion people contracting trichomoniasis annually. Trichomoniasis is the most common parasitic infection in the US and has an annual incidence estimated at 5 million cases. The frequency of infection and an increase in the number of drug resistant clinical isolates of T. vaginalis underscore the need to develop new chemotherapeutic strategies and drugs that eliminate the parasite. Resistance to the only drugs licensed for therapy, the 5-nitroimidazole (5NI) drugs metronidazole (Mz) and tinidazole (Tz), occurs in approximately 5% of cases of trichomoniasis. 5NIs are prodrugs that are activated by selective reduction inside anaerobic microbes, a step that is critical for killing, but also responsible for resistance when the microbe loses its capacity to reduce 5NIs. Upon activation, 5NIs kill the cell by forming covalent, inactivating adducts with essential target molecules, which are poorly defined. The long-term goal of the research proposed here is to define microbial targets of 5NI drugs that are essential for the survival of T. vaginalis and leverage this information to develop inhibitors of protein targets that are effective in the treatment of drug resistant trichomoniasis. The proposed studies have five Specific Aims. The first two aims will be achieved during the R21 phase and the latter three during the R33 phase. In Aim 1, we will identify nitro drug targets in model T. vaginalis strains using a strategy that employs click chemistry and mass spectrometry. In Aim 2, we will determine which protein targets are essential for T. vaginalis viability using reverse genetics to test whether elimination of the gene is lethal. After transitioning to the R33 phase, in Aim 3 we will identify nitro drug targets that are common to a broad range of geographically and genetically diverse T. vaginalis clinical isolates. In Aim 4, we will determine whether selected, common targets identified in Aim 3 are required for parasite viability. In Aim 5 we will develop inhibitors for the most promising nitro drug targets and examine inhibitor activity in vitro and in vivo. In the end, we aim to have validated novel drug targets and developed new inhibitors against these targets as candidates for effective treatment of Mz resistant trichomoniasis. The proposed research will also pave the way for using the same approach to identify and validate new antimicrobials to treat other anaerobic parasitic infections, such as giardiasis and amoebiasis, for which Mz is the primary drug used for therapy.